Composition Comprising Various Proteorhodopsins and/or Bacteriorhodopsins and Use Thereof

ABSTRACT

The present invention provides a solid material comprising an immobilized mixture of two or more proteorhodopsins, two or more bacteriorhodopsins, or one or more bacteriorhodopsin and one or more proteorhodopsins. The proteorhodopsins are selected from the group consisting of all-trans-retinal-containing proteorhodopsins and retinal analog-containing proteorhodopsins; all of which have absorption spectra that do not overlap. The bacteriorhodopsins are selected from the group consisting of all-trans-retinal-containing bacteriorhodopsins and retinal analog-containing bacteriorhodopsins; all of which have absorption spectra that do not overlap. The present invention also provides an optical information carrier, such as an optical data storage material and a fraud-proof optical data carrier, comprising the above-described solid material and a substrate selected from the group consisting of glass, paper, metal, fabric material, and plastic material, wherein said solid material is deposited on said substrate. The present invention further provides security ink comprising one or more hydrophilic polymers and a mixture of various photochromic materials.

FIELD OF INVENTION

The present invention relates to a solid material having an immobilizedmixture of various photochromic materials having absorption spectra thatdo not overlap significantly. The various photochromic materials areall-trans-retinal-containing proteorhodopsins, retinal analog-containingproteorhodopsins, all-trans-retinal-containing bacteriorhodopsins and/orretinal analog-containing bacteriorhodopsins. Particularly, theinvention relates to use of a mixture of various photochromic materialsas optical data storage materials, fraud-proof optical data carriers,and security ink.

BACKGROUND OF THE INVENTION

Bacteriorhodopsin (BR) is a retinal protein molecule found in thephotosynthetic system of a salt-marsh bacterium called Halobacteriumsalinarium. The BR molecules are located in the cell membrane, forming a2D protein-lipid array, commonly called the purple membrane. The use ofphotochromic proteins like bacteriorhodopsin (BR) for optical datastorage has been considered promising.

Proteorhodopsins (PRs) are distantly related to bacteriorhodopsin (BR)(22-24% sequence identity). Proteorhodopsins are integral membraneproteins; they are isolated from uncultivated marine eubacteria andfunction as light-driven proton pumps. Upon absorption of light by theall-trans-retinal co-factor, proteorhodopsin goes through a photocyclewith a number of intermediates. It is believed that upon excitation ofthe proteorhodopsin molecule by light stimulation, aproteorhodopsin/retinal complex is excited to an unstable intermediateenergy state. Proteorhodopsin progresses through a series of unstableenergy states that can vary in terms of energy plateaus orintermediates, e.g., an “M-like state” or “M-state”, a “K-like state” or“K-state”, an (“N-like state” or “N-state”, or an “O-like state” or“O-state”. Subsequently, the complex reverts to a more stable basalstate concomitant with transport of a proton.

Béjà, et al. (Science 289:1902-6, 2000) disclose the cloning of aproteorhodopsin gene from an uncultivated member of the marineγ-proteobacteria (i.e., the “SAR86” group). The proteorhodopsin wasfunctionally expressed in E. coli and bound all-trans-retinal to form anactive light-driven proton pump.

Béjà, et al. (Nature 411:786-9, 2001) disclose the cloning of overtwenty variant proteorhodopsin genes from various sources. Theproteorhodopsin variants appear to belong to an extensive family ofglobally distributed proteorhodopsin variants that maximally absorblight at different wavelengths.

Dioumaev, et al. (Biochemistry, 42: 6582-6587 (2003)) disclose usingproteorhodopsin-containing membrane fragments encased in polyacrylamidegel for flash photolysis and measurements of absorption changes in thevisible range.

U.S. Pat. No. 5,235,076 (Asato) discloses azulenic retinoid compoundsand therapeutic compositions. The compositions are useful in treatingdermatological disorders such as acne and psoriasis.

U.S. Pat. No. 4,896,049 (Ogawa) discloses various synthetic analogs ofretinal, which have different absorption wavelengths. The syntheticretinal analogs disclosed in Ogawa are incorporated herein by reference.

Khodonov, et al. (Sensors and Actuators B 38-39:218-221 (1997)) describemodified bacteriorhodopsin by replacing the natural bacteriorhodopsinchromophore, all-trans-retinal, with its analogs. The retinal analogsdisclosed in Khodonov are incorporated herein by reference.

Imai, et al. (Photochemistry and Photobiology, 70: 111-115 (1999))disclose that azulenic retinal analogs failed to yield a red-shiftedvisual pigment analog, whereas the 9-cis isomers of the polyenals3-methoxy-3-dehydroretinal and 14F-3-methoxy-3-dehydroretinal yieldediodopsin pigment analogs at 663 and 720 nm.

U.S. Pat. No. 6,483,735 (Rentzepis) discloses a three- orfour-dimensional radiation memory that serves to store multiple binarybits of information in the same physical volumes of each of amultiplicity of addressable domains in each of potentially multiplelayers within the entire volume of a planar disc, or in a random-accessvolume radiation memory. The storage of multiple information bits withinthe same addressable domains is done by the co-location of severaldifferent florescent chemical compounds in the volume of each suchdomain; the florescent chemical compounds are not rewriteable.

U.S. Pat. Nos. 5,470,690 and 5,346,789 (Lewis) disclose a stable,image-retaining, optically switchable film containing bacteriorhodopsinobtained from Halobacterium Halobium (currently known as Halobacteriumsalinarum) in a high-pH polyvinyl alcohol solution for an optical memoryfor data storage.

Gourevich, I. et al. (Chemical Materials, Multidye NanostructuredMaterial for Optical Data Storage and Security Labeling (2004)) disclosea polymer nanocomposite for three-dimensional optical data storage andsecurity labeling using visible and near-IR fluorescent dyes. The datais written via selective photobleaching of the fluorescent dyes, whichare not rewriteable.

Optical data storage has the potential to revolutionize the computerindustry, since optical data storage provides both a very high storagecapacity and rapid reading and writing of data. Additionally, opticalsignal processing could be used in a highly parallel fashion for patternrecognition, which is difficult to do with the current computingtechnologies. A functional optical material with low light scattering,large data storage capacity, and rewriteable capacity is required forthese applications to succeed.

Documents like banknotes, checks, identity cards, etc. often incorporatesecurity features to make them difficult to copy or counterfeit. Most ofthese are based on either using special paper with security featureslike watermarks incorporated during paper manufacturing, or printinghairline patterns that are difficult to copy. However, such features arepermanently visible and do not meet sophisticated security requirements.

There are needs for optical information carriers that can be producedefficiently and economically and have low background noise (crosstalk),large data storage capacity, and rewriteable capacity. Such opticalinformation carriers are effective as optical data storage material orfraud-proof optical data carriers.

SUMMARY OF THE INVENTION

The present invention provides a solid material comprising animmobilized mixture of photochromic materials that have absorptionspectra that do not overlap significantly among each other. Thephotochromic materials comprise two or more proteorhodopsins, two ormore bacteriorhodopsins, or one or more bacteriorhodopsin and one ormore proteorhodopsins. The proteorhodopsins are selected from the groupconsisting of all-trans-retinal-containing proteorhodopsins and retinalanalog-containing proteorhodopsins. The bacteriorhodopsins are selectedfrom the group consisting of all-trans-retinal-containingbacteriorhodopsins and retinal analog-containing bacteriorhodopsins. Thesolid material preferably comprises one or more hydrophilic polymersthat are capable of forming a homogeneous phase with said photochromicmaterials prior to solidification to a solid form.

The present invention provides an optical information carrier comprisingthe solid material as described above, wherein data are writtendifferentially by actinic light (writing light) of various wavelengthsand/or optical signals are read differentially by reading light ofvarious wavelengths. The optical signals can be read differentially bydetermining the decrease of B-state molecules of each photochromicmaterial. Alternatively, the optical signals can be read differentiallyby determining the light absorbance at maximum absorption wavelength ofthe M-state, or other excited state, by each photochromic material. Thevarious photochromic materials provide non-destructive writing andreading of data and are capable of being reused. The optical informationcarrier further comprises a substrate selected from the group consistingof glass, paper, metal, fabric material, and plastic material, whereinthe solid material is deposited on said substrate. The opticalinformation carrier of the present invention is, for example, afraud-proof optical data carrier or an optical data storage material.

The present invention further provides security ink comprising differentproteorhodopsins and/or bacteriorhodopsins as described above and one ormore hydrophilic polymers, wherein said different proteorhodopsinsand/or bacteriorhodopsins and the hydrophilic polymers form ahomogeneous liquid phase, said ink solidifies or dries after applicationonto a surface, thereby immobilizing said various photochromic materialsonto a specific location where the ink is applied.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the temporary data storage spectra of a mixture ofbacteriorhodopsin and proteorhodopsin immobilized in a transparentmatrix. The spectra were recorded in sequence as described. Spectrum 1was taken after the mixture was illuminated with a violet light (400nm). Spectrum 2 was taken after the mixture was illuminated with a greenlight (510 nm). Spectrum 3 was taken after the mixture was illuminatedwith a violet light (400 nm), followed by a red light (640 nm).

FIG. 2 shows the temporary data storage spectra of a mixture ofbacteriorhodopsin and proteorhodopsin immobilized in a transparentmatrix. The spectra were record in sequence following those described inFIG. 1 as described. Spectrum 4 was taken after the mixture wasilluminated with a green light (510 nm). Spectrum 5 was taken after themixture was illuminated with a violet light (400 nm). Spectrum 6 wastaken after the mixture was illuminated with a violet light (400 nm),followed by a red light (640 nm).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “actinic light” refers to radiant energy,especially in the visible and ultraviolet spectral regions, which canproduce photochromic changes in a photochromic material.

As used herein, the term “apoprotein” refers to the protein part of aconjugated protein. A “proteorhodopsin or bacteriorhodopsin apoprotein”refers to the proteorhodopsin or bacteriorhodopsin protein itselfwithout the all-trans-retinal or retinal analog.

As used herein, the term “azulenic retinoid compound” refers to acompound having azulenic group attached to a modified or non-modifiedall-trans-retinal backbone.

As used herein, the term “basal state” or “B-state” or “B-like state”refers to the basal state of the photocycle of a proteorhodopsinmolecule or a bacteriorhodopsin molecule without light excitation. Theterm “M-state” or “M-like state” refers to an excited spectral state ina photocycle as compared with the basal state.

As used herein, “photochromic” refers to having the capability to changecolor upon exposure to radiant energy (as light).

As used herein, the term “retinal analog” refers to a compound thatreplaces all-trans retinal and is capable of coupling with theapoprotein of a proteorhodopsin or a bacteriorhodopsin.

The present invention provides a solid material comprising animmobilized mixture of various photochromic materials, wherein saidvarious photochromic materials all have absorption spectra that do notoverlap significantly.

The solid material of the present invention comprises one or morehydrophilic polymers that are capable of forming a homogeneous phasewith said various photochromic materials prior to solidification to asolid form. The solid material that contains a mixture of variousphotochromic materials is useful as optical information data carriersuch as an optical data storage material and fraud-proof optical datacarrier. The various photochromic materials, which have absorptionspectra that do not overlap significantly, provide an increased capacityof optical data storage and allow for parallel processing. The solidmaterial is useful in storing (writing) optical data. The material iscapable of retaining data, permits nondestructive detection (reading) ofsuch data, and is reuseable after optical erasure of data.

In one embodiment, the photochromic material that has changed color hasthe ability to return to the original color. Return to the originalcolor by the photochromic material can be spontaneous or caused byre-exposure to radiant energy.

Various photochromic materials of the present invention include variousproteorhodopsins and/or bacteriorhodopsins; all of which have absorptionspectra that do not overlap significantly. By “not overlapsignificantly,” it is meant that a particular wavelength can be selectedsuch that the absorbance (optical density) at that wavelength of oneproteorhodopsin or bacteriorhodopsin is at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, or atleast 10 times higher than the absorbance of the other proteorhodopsinor bacteriorhodopsin under the same conditions (e.g. temperature). Forexample, at a selected wavelength such as 600 nm, if photochromiccomposition X has an absorbance of 1.0 OD, and photochromic compositionY has an absorbance equal to or less than 0.5, preferably 0.33,preferably 0.2, more preferably 0.1 OD, then the absorbance spectra ofphotochromic compositions X and Y do not overlap significantly.

In one embodiment of the invention, various photochromic materialscomprise two or more (e.g. three, four, five, six, seven, eight, etc.)proteorhodopsins. In another embodiment of the invention, variousphotochromic materials comprise two or more bacteriorhodopsins (e.g.three, four, five, six, seven, eight, etc.). In yet another embodimentof the invention, various photochromic materials comprise one or morebacteriorhodopsins and one or more proteorhodopsins. In the presentinvention, the proteorhodopsins are selected from the group consistingof all-trans-retinal-containing proteorhodopsins and retinalanalog-containing proteorhodopsins. The bacteriorhodopsins are selectedfrom the group consisting of all-trans-retinal-containingbacteriorhodopsins and retinal analog-containing bacteriorhodopsins.

Proteorhodopsins

Proteorhodopsin is a trans-membrane protein with a structure of sevenlipid membrane-spanning α-helices that form a generally cylinder shapedchannel. When folded correctly and supplied with all-trans-retinal, theseven α-helices of proteorhodpsin are arranged as a cage surrounding theall-trans-retinal. One advantage of using proteorhodopsins in an opticalinformation carrier is that proteorhodopsins can be functionallyexpressed in E. coli to produce a large quantity (grams or kilograms) ofprotein economically and efficiently. The proteorhodopsin-expressingcells are lysed and the pellets containing the membrane fraction arecollected. The proteorhodopsin protein can be further extracted from themembrane by detergent solubilization. Either the membranes or fragmentsof membranes that contain proteorhodopsins, or the purifiedproteorhodopsin proteins can be used as an optical information carriersuch as an optical data storage material or a fraud-proof optical datacarrier.

When using proteorhodopsins as an optical data storage material, it isdesirable to immobilize detergent-solubilized proteorhodopsins to avoidlight scattering. In one embodiment of the invention,detergent-solubilized and membrane-free proteorhodopsins are used.Detergent-solubilized proteorhodopsins are usually in the form of amonomer, and sometimes in the form of an oligomer (dimer, trimer,tetramer, pentamer, or hexamer). Individual proteorhodopsin monomers areabout 5 nm in size; such small size does not cause scattering of lightin the visible range. The monomeric or oligomeric stability ofproteorhodopsin makes it desirable as a component of an optical datastorage material without having the problem of a μm-sized particle thatscatters light. Additionally, the small size of the individualproteorhodopsin monomers makes it easier to obtain a uniform proteindistribution in the optical data storage material.

Different from bacteriodopsins, proteorhodopsins are stable in itsmonomeric or oligomeric state for at least one month at roomtemperature, or one year at 4° C. The term “stable” refers to thatproteorhodopsin does not change its spectral property significantly(less than 30 nm in maximum absorption wavelength) and is able toproduce a photocycle upon excitation by light that includes a transitionfrom the basal state to the M-state.

The basal absorption maxima of all-trans-retinal-containingproteorhodopsin variants are in general between 480 nm and 550 nm, oftenbetween 488 and 526 nm. (Man, et al. Embo J. 22:1725-1731 (2003))

The absorption maxima of the M-state of proteorhodopsins in general arebetween 350 nm and 450 nm, often about 410 nm. The M-state isdistinguished from other identified spectral states, the K-, N- andO-like states, which all have red-shifted absorption spectra (e.g. >530nm) compared with the basal state.

When a proteorhodopsin molecule is exposed to actinic light of anexcitation wavelength, it is excited to an activated M-state and changesto a yellow color. The color is reverted to its basal color eitherspontaneously with time or by exposing the material to a second light.For example, the proteorhodopsin-containing material is excited by ayellow light or a green light to change color from red or purple toyellow; the color change is erased spontaneously or by illuminating thematerial with purple or blue light. The excitation and erasing cycle canbe repeated many times, thus, the proteorhodopsin molecule is re-usable.

Proteorhodopsins useful for the present invention can be derived fromany naturally occurring proteorhodopsin. Various natural nucleic acidsequences, encoding various natural proteorhodopsins, have been obtainedfrom naturally occurring members of the domain bacteria. Such membersinclude marine bacteria, such as bacteria from the SAR86 group. Thenatural nucleic acid sequences of proterhodopsins are cloned and thenatural form of proteorhodopsins is expressed. There are many naturalforms of proteorhodopsins; including those derived from marine bacteriaand those derived from non-marine bacteria; all of which can be used forthe present invention.

For example, natural forms of proteorhodopsins include Hot75 ml,Bac31A8, Bac40E8, Bac41B4, Bac64A5, Hot0m1, Hot75m3, Hot75m4, Hot75m8,MB0m1, MB0m2, MB20m2, MB20m5, MB20 m12, MB40 ml, MB40m5, MB100m5,MB100m7, MB100m9, MB100m10, PalB1, PalB2, PalB5, PalB7, PalB6, PalB8,PalE1, PalE6, PalE7, MED 26, MED27, MED36, MED101, MED102, MED106,MED25, MED202, MED204 MED208, REDA9, REDB9, REDF9, RED19, RED2, RED23,RED27, RED30, RED4, RED5, REDr6a5a14, REDr6a5a6, REDr7_(—)1_(—)4,REDs3_(—)7, REDr7_(—)115, REDs3_(—)15, medA1r8ex6, REDr7_(—)1_(—)16,medA15r11b9, medA15r9b5, medA15r8b3, medA15r11b3, medA15_r8_(—)1,medA17R9_(—)1, medA15r8b9, medA19_R8_(—)16, medA19_R8_(—)19,medA17_R8_(—)6, medA15r9b7, medA15R8_(—)3, medA15r10b5, medA19_r9_(—)9,medA15_r8ex7, medA19_R8_(—)20, medA15_R8ex9, medA15_r9_(—)3,medA17_r8_(—)15, medA17_r8_(—)11, medA15r8b8, medA15r8ex4, ANT32C12 PRand HOT2C01 PR. See Baja, et al., Nature 411:786-9 (2001); Man, et al.,EMBO J., 22:1725-1731 (2003); and Sabehi, et al., Environ. Microbiol.,5: 842-9 (2003). The nucleotide and amino acid sequences of the abovevarious proteorhodopsins have been deposited with Genbank underaccession numbers AF349976-AF350003, AF279106, AY210898-AY210919,AY250714-AY250741, AY372453 and AY372455. In addition, Venter, et al.(Science 304: 66-74 (2004)) recently have reported 782 new rhodopsinanalogs, most of which are proteorhodopsins, found in the Sargasso Sea.The proteorhodopsins described in the above references are suitable forthe present invention.

Proteorhodopsins useful for the present invention can also be derivedfrom any non-naturally occurring proteorhodopsins, such asproteorhodopsin mutants. The term “proteorhodopsin mutant” refers to aproteorhodopsin comprising one or more mutations that insert, delete,and/or substitute one or more amino acid residues and/or nucleotidesfrom the natural sequences of proteorhodopsins. For example, thenucleotide sequence can be altered by a substitution of a differentcodon that encodes the same or a functionally equivalent amino acidresidue within the sequence, thus producing a silent change. Forexample, an amino acid residue within the sequence can be substituted byanother amino acid of a similar polarity, or a similar class. Non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, glycine and methionine. Polarneutral amino acids include serine, threonine, cysteine, tyrosine,asparagine, and glutamine. Positively charged (basic) amino acidsinclude arginine, lysine, and histidine. Negatively charged (acidic)amino acids include aspartic and glutamic acid.

Proteorhodopsin mutants useful for the present invention, for example,include the amino acid sequence of Bac31A8H75K, Bac31A8H75N,Bac31A8H75Q, Bac31A8 E108Q, Bac31 A8 D97N, Hot75 ml H77K, Hot75 ml H77N,Hot75 ml H77Q, Hot75 ml H77E, Hot75 ml H77D, Hot75 ml H77W, Hot75 mlR96A, Hot 75 ml E110Q, Hot75 ml D99N, Hot75 ml R96E, and Hot75 ml R96Q.In which, Bac31A8H75K means that the 75 amino residue of the naturallyoccurring Bac31A8 is mutated from histine to lysine. Proteorhodopsinmutants have been disclosed in the co-pending U.S. ApplicationPublication No. 2005-0095605; which is incorporated herein by referencein its entirety.

Bacteriorhodopsins

Bacteriorhodopsin (BR) is an all-trans-retinal-containing proteinmolecule found in the photosynthetic system of a salt-marsh bacteriumcalled Halobacterium salinarium. BR-based optical films have been workedon for the past two decades, but by themselves, these films do not havethe required properties to make them commercially viable for datastorage applications. One of the problems with the BR-based films isthat BR forms 0.2-1 μm sized protein-lipid patches. If BR is extractedfrom these patches to form a monomeric protein, it becomes unstable andis inactivated in a few days. The problem with using these BR patches inoptical films is that the patches are approximately the same size as thewavelength of the light used to interface with the film, which resultsin significant light scattering during read and write cycles, therebyincreasing noise and degrading the performance of the film.Additionally, the BR patches tend to stick to each other, which resultin uneven distribution of the BR protein in the film, and furtherdegrade the performance of BR-based optical films.

Another disadvantage of BR in comparison with PR is that BR is expensiveto produce in a large quantity. BR has to be expressed in its naturalorganism H. salinarum for it to be fully functional (Dunn, et al., JBiol Chem, 262: 9246-9254 (1987); Hohenfeld, et al., FEBS Lett, 442:198-202 (1999)). H. salinarum grows very slowly, gives a low celldensity and requires the presence of large amounts of salt in the growthmedium. The low productivity of H. salinarum and the need for expensivecustom-made fermentation and recovery equipment that can tolerate thehigh salt growth medium result in high cost of BR production.

Nonetheless, bacteriorhodopsin has a unique maximum absorbancewavelength of 590 nm, which is different from those of mostproteorhodopsins, and is thus useful as a component of the variousphotochromic materials. BR molecules are useful when combined in amaterial consisting of PR molecules.

The basal absorption maxima of all-trans-retinal-containingbacteriorhodopsins are in general 590 μm, without any significantvariation. All-trans-retinal-containing bacteriorhodopsins all have thesimilar purple color. The absorption maxima of the M-state ofbacteriorhodopsins in general are about 410 nm.

When bacteriorhodopsin is exposed to light of excitation wavelength, itis excited to an activated M-state and changes to yellow color. Thecolor is reverted to its basal color either spontaneously with time orby exposing the material to a second light. For example, thebacteriorhodopsin-containing material can be excited by a green light tochange color from purple to yellow; the color change is erasedspontaneously or by illuminating the material with blue light.

Bacteriorhodopsins useful for the present invention can be derived fromany naturally occurring bacteriorhodopsins. Bacteriorhodopsins usefulfor the present invention can also be derived from any non-naturallyoccurring bacteriorhodopsins, such as bacteriorhodopsin mutants. Theterm “bacteriorhodopsin mutant” refers to a bacteriorhodopsin comprisingone or more mutations that insert, delete, and/or substitute one or moreamino acid residues and/or nucleotides from the natural amino or nucleicacid sequence of bacteriorhodopsin. For example, the nucleotide sequencecan be altered by a substitution of a different codon that encodes thesame or a functionally equivalent amino acid residue within thesequence, thus producing a silent change. For example, an amino acidresidue within the sequence can be substituted by another amino acid ofa similar polarity, or a similar class. Non-polar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, glycine and methionine. Polar neutral aminoacids include serine, threonine, cysteine, tyrosine, asparagine, andglutamine. Positively charged (basic) amino acids include arginine,lysine, and histidine. Negatively charged (acidic) amino acids includeaspartic and glutamic acid.

Bacteriorhodopsin mutants useful for the present invention include:BR-D85N and BR-D96N, (Hampp, Chem. Rev., 100:1755-1776 (2000)), BR-T90V,BR-D115L, BR-V49A (Dioumaev, et al., Biochemistry 41(17):5348-58(2002)), BR-E194C (Balashov, et al., Biochemistry 36:8671-8676 (1997)),BR-E194Q and BR-E204Q (Dioumaev, et al., Biochemistry 37:2496-2506(1998)), BR-R82A and BR-D85E (Subramaniam, et al., Proc. Natl. Acad.Sci. U.S.A., 87:1013-1017 (1990)), BR-D85A, BR-D85N, BR-D85E, BR-D212N,BR-D212E, BR-R82A, BR-R82Q, BR-D115A, BR-D115N, BR-D115E, BR-D96A,BR-D96N, BR-D96E (Otto, et al., Proc. Natl. Acad. Sci. U.S.A.87:1018-1022 (1990)), BR-E204Q, BR-E204D, BR-L93M, BR-L93T, BR-L93S(Kandori, et al., Biochemistry 36:5134-5141 (1997)), BR-V49A (Brown, etal., Biochemistry 33:12001-12011 (1994)), BR-L93A (Delaney, et al., J.Phys. Chem. B., 101:5619-5621 (1997)), as well as other possiblebacteriorhodopsin mutants. In which, BR-D85N means that the 85 aminoacid residue of the naturally occurring bacteriorhodopsin is mutatedfrom aspartic acid (D) to asparagine (N).

Retinal Analogs

Various retinal analogs are useful in the present invention. In oneembodiment, the retinal analog is an azulenic retinoid compound. Inanother embodiment, the retinal analog is a compound that isstructurally similar to all-trans-retinal. Aproteorhodopsin/bacteriorhodopsin apoprotein and a retinal analog form aphotochromic material having spectral properties different from acorresponding photochromic material comprising the sameproteorhodopsin/bacteriorhodopsin apoprotein and all-trans-retinal.

In one embodiment of the application, aproteorhodopsin/bacteriorhodopsin apoprotein and a retinal analog form aphotochromic material, whose absorbance spectrum does not overlapsignificantly from the absorbance spectrum of a correspondingphotochromic material comprising the sameproteorhodopsin/bacteriorhodopsin apoprotein and all-trans-retinal underthe same condition (e.g. temperature). In another embodiment of theapplication, a proteorhodopsin/bacteriorhodopsin apoprotein and retinalanalog form a photochromic material that yields a red-shifted visualchromophore compared with a photochromic material comprising the sameproteorhodopsin/bacteriorhodopsin apoprotein and all-trans-retinal underthe same condition (e.g. temperature). The changes in spectralproperties provide the use of multiple wavelengths of light to increasecapacity of optical data storage and allow parallel processing.

The structure of all-trans-retinal is shown as following:

Retinal analogs useful for the present invention include azulenicretinoid compounds of Formula I:

wherein R′, R″ and R′″ are each independently H, C₁₋₄ straight chainalkyl, or CIA branched chain alkyl,n is an integer from 1 to 4;X_(a) and X′_(b) are each independently H, C₁₋₄ alkyl, F, Cl or CF₃;Y is absent, or Y is a para-, meta-, or ortho-phenyl; and

Z is CHO.

In one embodiment of the invention, R′, R″ and R′″ are independently H,methyl, isopropyl. Preferably, R′=R′″=methyl, R″=isopropyl.In a preferred embodiment of the invention, Y is absent.The preparation of azulenic retinoid compounds is disclosed, forexample, in U.S. Pat. No. 5,235,076 (Asato), which is incorporatedherein by reference in its entirety.Specific examples of Formula I that are useful for the present inventioninclude the following compounds:

The retinal analogs useful for the present invention also include thefollowing non-azulenic compounds that are structurally similar toall-trans-retinal:

P-dimethyl aminocinnamaldehyde (DMCA)

DMCA is commercially available, for example, from Fluka AG (Buchs,Switzerland) through Sigma-Aldrich (St. Louis, Mo.), The Lab Depot, Inc.(Alpharetta, Ga.) and The Science Lab.Com (Kingwood, Tex.).

The retinal analog-containing proteorhodopsin can be convenientlyprepared by expressing proteorhodopsin in the presence of the analog ina host cell. E. coli, for example, is an effective host cell because itdoes not produce all-trans-retinal. Other host cells, in which thesynthetic pathway of all-trans-retinal is blocked, can also be effectivehost cells for preparing the retinal analog-containing proteorhodopsins.The analog is added to the cell culture and inserted into theproteorhodopsin protein during host cell growth and proteorhodopsinexpression.

The retinal analog-containing bacteriorhodopsin, on the other hand,cannot be prepared conveniently by adding the analog during theexpression of bacteriorhodopsin in its host cell H. salinarum.all-trans-retinal is produced by H. salinarum, and theall-trans-retinal-containing bacteriorhodopsin is formed during theexpression of bacteriorhodopsin. In order to prepare retinalanalog-containing bacteriorhodopsin, the all-trans-retinal needs to beremoved from the all-trans-retinal-containing bacteriorhodopsin, thenthe retinal analog is added to the bacteriorhodopsin apoprotein to forma complex of bacteriorhodopsin and retinal analog.

A Solid Material Comprising a Mixture of Immobilized Proteorhodopsinand/or Bacteriorhodopsin

The present invention provides a solid material comprising animmobilized mixture of one or more bacteriorhodopsins and one or moreproteorhodopsins; preferably, all of the bacteriorhodopsins andproteorhodopsins have absorption spectra that do not overlapsignificantly. The present invention also provides a solid materialcomprising an immobilized mixture of two or more proteorhodopsins, whichhave absorption spectra that do not overlap significantly. The presentinvention additionally provides a solid material comprising animmobilized mixture of two or more bacteriorhodopsins, which haveabsorption spectra that do not overlap significantly. In the above solidmaterials, the proteorhodopsins are selected from the group consistingof all-trans-retinal-containing proteorhodopsins and retinalanalog-containing proteorhodopsins, and the bacteriorhodopsins areselected from the group consisting of all-trans-retinal-containingbacteriorhodopsins and retinal analog-containing bacteriorhodopsins.

The solid material of the present invention preferably comprises one ormore hydrophilic polymers that are capable of forming a homogeneousphase with proteorhodopsins and/or bacteriorhodopsins prior tosolidification to a solid form such that the proteorhodopsins and/orbacteriorhodopsins are evenly distributed in the solid. By “homogeneous”is meant that the proteorhodopsins and/or bacteriorhodopsins and thehydrophilic polymer or its precursor form a uniform structure orcomposition throughout the mixture. As used herein, by “immobilized” ismeant that proteorhodopsin/bacteriohodopsin is not mobile, and is fixedwithin the material. The interaction between proteorhodopsin and thematerial can be covalent or non-covalent. For example,proteorhodopsin/bacteriohodopsin can be physically entrapped within thematerial. Proteorhodopsin can also bind to the material by electrostaticcharges, H-bond, hydrophobic, hydrophilic, or van der Waals interaction.By immobilization, the proteorhodopsin/bacteriohodopsin molecules arefixed and do not diffuse or diffuse very slowly within the solidmaterial, such that an optical signal is not lost by diffusion of theproteorhodopsin molecules.

The hydrophilic polymers produce a non-opaque or optically transparentsolid material, which allows efficient light excitation of thephotochromic material contained therein.

Hydrophilic polymers suitable for this invention include silica sol gel,gelatin, polyacrylamide, acacia, agar, calcium carrageenan, calciumalginate, sodium alginate or other salts of alginic acid, algin,agarose, collagen, methyl cellulose, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylic acid, partially cross-linked polyacrylicacid, hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, polyethylene oxide, pectin and mixturesthereof.

Vinyl polymers and derivatives thereof are also useful in the presentinvention. Polyvinyl alcohol (PVA), is defined as a homopolymer orcopolymer, in which vinyl acetate is a starting monomer unit and inwhich most or all (70-100%) of the acetate moieties are subsequentlyhydrolyzed to alcohol moieties. Other vinyl polymers useful in thepresent invention include, but are not limited to, polyvinyl acetate andpolyvinyl pyrrolidone. Copolymers such as PVA-methylmethacrylatecopolymer may also be used in the present invention. PVA is commerciallyavailable in a wide range of molecular weights, viscosities and varyingdegrees of hydrolysis from the polyvinyl acetate precursor.

Other polymers useful for this invention include polymers that formhydrogels such as Carbopol®, acidic carboxy polymers; Cyanamey-Opolyacrylamides; cross-linked indene-maleic anhydride polymers, Polyox®polyethylene oxide polymers; starch graft copolymers; Aqua-Keepsoacrylate polymer polysaccharides composed of condensed glucose unitssuch as diester cross-linked polyglucan, and the like. Representativepolymers that form hydrogel are shown in U.S. Pat. Nos. 3,865,108;4,002,173; 4,207,893; and in Handbook of Common Polymers, by Scott andRoff, published by the Chemical Rubber Company, Cleveland, Ohio.

A solid material containing an immobilized mixture of variousproteorhodopsin(s) and/or bacteriorhodopsin(s) in a hydrophilic polymeror in a mixture of hydrophilic polymers can be prepared by the steps offirst mixing a hydrophilic polymer or its precursor with variousproteorhodopsin(s) and/or bacteriorhodopsin(s) in water or an aqueousbuffer to form a homogeneous solution, then solidifying the polymer,wherein the various proteorhodopsin and/or bacteriorhodopsin moleculesare immobilized in the polymer. The solidification of the polymer iscarried out by drying, cooling, curing, or polymerization.

For example, a polyvinyl alcohol material containing an immobilizedmixture of various proteorhodopsin and/or bacteriorhodopsin moleculescan be prepared by the method comprising the steps of: (a) mixingpolyvinyl alcohol, water or a buffer having pH between about 3-12, andvarious proteorhodopsin and/or bacteriorhodopsin molecules to form asolution; (b) spreading the solution on the surface of a solid; and (c)drying the solution to form a polyvinyl alcohol material containingimmobilized various proteorhodopsin and/or bacteriorhodopsin molecules

A polyacrylamide material that contains immobilized variousproteorhodopsin and/or bacteriorhodopsin molecules can be prepared bythe method comprising the steps of (a) mixing acrylamide, bisacrylamide,various proteorhodopsin and/or bacteriorhodopsin molecules, and one ormore polymerization initiators in water or a buffer having pH between3-12; and (b) polymerizing acrylamide gel; whereby the variousproteorhodopsin and/or bacteriorhodopsin molecules are immobilizedwithin the polyacrylamide gel matrix. The polymerization initiatorscommonly used include ammonium persulfate andN,N,N′,N′-tetramethylethylenediamine (TEMED). Alternatively, the methodcomprises the steps of (a) mixing acrylamide, bisacrylamide, variousproteorhodopsin and/or bacteriorhodopsin molecules, and one or moreUV-activated free radical generators in water or a buffer having pHbetween 3-12; and (b) exposing the mixture to UV light to polymerizeacrylamide gel. The UV-activated free radical generators includeriboflavin and TEMED (used together), 2,2-Dimethoxy-2-phenylacetophenone (DMPA), and those described in the SE96047-3 patent.

Sol-gels that contain immobilized various proteorhodopsin and/orbacteriorhodopsin molecules can be prepared by the method comprising thesteps of: (a) adding to a silane precursor an acidic solution having pH1.5-4 to hydrolyze the silane precursor to form silicate sol; (b) addingto the silicate sol an aqueous solution containing variousproteorhodopsins and/or bacteriorhodopsins at pH about 5-9; and (c)incubating (b) to form a gel; whereby the various proteorhodopsin and/orbacteriorhodopsin molecules are immobilized within the sol gel matrix.The silane precursors include tetraalkylorthosilicate,alkyltrialkoxysilane, aryltrialkoxysilane, dialkyldialkoxysilane,diaryldialkoxysilane, alkali metal silicate, polyol silicate, polyolsiloxane, poly(methyl silicate), and alcohol-free poly(silicic acid).Preferred silane precursors are tetraaylorthosilicate andpoly(glyceryl)silicate.

Gelatin that contains immobilized various proteorhodopsins and/orbacteriorhodopsins can be prepared by the method comprising the stepsof: (a) heating and dissolving gelatin in water or a buffer to form ahomogeneous aqueous gelatin solution; (b) cooling the gelatin solutionto about 39-45° C.; (c) mixing the cooled gelatin solution with variousproteorhodopsins and/or bacteriorhodopsins; and (d) incubating (c) toform a gel; whereby the various proteorhodopsins and/orbacteriorhodopsins are immobilized within the gelatin gel matrix.

The solid material of the present invention contains an immobilizedmixture of various proteorhodopsin and/or bacteriorhodopsin molecules.The various proteorhodopsin and/or bacteriorhodopsin molecules arepre-mixed prior to solidification to a solid form. Because the variousproteorhodopsin and/or bacteriorhodopsin molecules are mixed in amolecular level, they are able to locate within the same addressabledomains. This provides an economic procedure to produce variousphotochromic materials, which can be independently written and read,within the same addressable domains.

TECHNICAL APPLICATION

The proteorhodopsins and/or bacteriorhodopsins of the present inventionhave many technical applications. For example, they can be incorporatedinto instruments or devices having photochromic applications,photoelectric applications, and/or phototransport applications.

Under photochromic applications, proteorhodopsins and/orbacteriorhodopsins can be used for its light absorption properties foroptical data storage, interferometry and/or photonics. Photochromicapplications include, but are not limited to, holographic film. Theretinal analog-containing proteorhodopsins can be used for optical datastorage devices, such as 2-D storage, 3-D storage, holographic storage,associative storage, or the like. The retinal analog-containingproteorhodopsins can be used in a device for information processing,such as optical bistability/light switching, optical filtering, signalconditioning, neural networks, spatial light modulators,phaseconjugation, pattern recognition, interferometry, or the like.

Under phototransport applications, proteorhodopsins and/orbacteriorhodopsins can be used for its light-induced proton transportacross a membrane, such as photovoltaic device. One such photovoltaicdevice is a light-driven energy generator comprising theproteorhodopsin, whereby light energy can be converted to chemicalenergy. The retinal analog-containing proteorhodopsins can also be usedin devices for ATP generation in reactors, desalination of seawater,and/or conversion of sunlight into electricity.

Proteorhodopsins and/or bacteriorhodopsins can also be used in devicesfor 2D harmonic generation, radiation detection, biosensor applications,or the like.

In one embodiment, the invention provides a material suitable for anoptical information carrier. Particularly, the material is suitable foroptical data storage material or fraud-proof optical data carrier.

In one embodiment, the invention provides a material suitable for thestorage and processing of optical information.

In one embodiment, the invention provides a material for use in storing(writing) optical data, the material being capable of retaining datawhile permitting nondestructive detection (reading) of such data, andbeing capable of reuse after optical erasure of data.

In one embodiment, the invention provides an optical information carriermaterial that is difficult for counterfeiters to mimic.

In one embodiment, the invention provides fraud-proof ink that changescolor upon exposing to light.

Optical Information Data Carrier

The present invention provides optical information carriers that can beproduced efficiently and economically and have low background noise(crosstalk), large data storage capacity, and rewriteable capacity. Suchoptical information carriers are effective as optical data storagematerial or fraud-proof optical data carriers.

The present invention provides an optical information data carriercomprising a solid material comprising an immobilized mixture of variousproteorhodopsins/bacteriorhodopsins as described above, wherein saidvarious proteorhodopsins/bacteriorhodopsins have absorption spectra thatdo not overlap significantly. The solid material can range in thicknessfrom a thinly deposited layer orders of magnitude larger in twodimensions than in the third dimension to a thickly cast object with alldimensions of comparable magnitude

The present invention provides an optical information carrier comprisinga solid material having a immobilized mixture of variousproteorhodposin/bacteriorhodopsin molecules and a substrate such asglass, paper, metal, fabric material, plastic material, wherein saidsolid material is deposited on said substrate. For example, thesubstrate is a disk, a card, or a document.

The optical information carrier of the present invention may be in theform of a thin film or membrane, which may be referred to as atwo-dimensional film, or may be in the form of a thick film which may bereferred to as a three-dimensional layer or block. The opticalinformation carrier so produced includes the mixture of variousproteorhodposin/bacteriorhodopsin molecules that can then beindependently exposed to light of different wavelengths to convert thevarious molecules from a basal state to a M state.

An alkaline pH such as pH 8-12 of the optical information carrier delaysthe decay of the light-induced M state, stabilizing the M-state andmaking it possible to imprint long-lasting optical images on thePR-containing film, even at room temperature. An alkaline pH iseffective for optical data storage because of longer lifetime ofM-state. The desirable length of time for data storage depends on theapplication and can vary between a few seconds, a few minutes, a fewhours, a few days, a few months, up to a few years. For fraud-proofapplication, short lifetime of M-state (a few seconds to severalminutes) is preferred.

The solid material containing an immobilized mixture of variousproteorhodopsin and/or bacteriorhodopsin molecules can be spread orsprayed on the surface of a document, a disk, and a card for use as anoptical data storage material. In one embodiment, the solid material canbe used in a volumetric data storage device or a holographic datastorage device. A volumetric data storage device is a type of a 3D datastorage device, in which a thickness of the data-recording material isdivided into a number of virtual planes that each contains stored data.A volumetric data storage device is therefore comparable to a stack of2D storage devices. A holographic data storage device is another type of3D data storage device; it uses the thickness of the film by recordingthe 3D interference pattern of a data carrying and a reference lightbeam.

Data are written in the optical information carrier of the presentinvention optically by exposing specific areas of the materialcontaining the mixture of photochromic molecules briefly to actiniclight of a particular wavelength. For example, the actinic light ispolychromatic yellow or green light (e.g. from a halogen lamp with a 450nm cut-on filter), monochromatic green light (e.g. from a green DiodePumped Solid State Frequency Doubled (DPSSFD) laser with a wavelength of532 nm). The exposed area becomes yellow, showing that particularphotochromic molecules in that area are excited by the light of acorresponding wavelength and converted to an activated M intermediate.This is the act of writing data to the solid material containing themixture of photochromic materials. Observing the color of the differentareas of the material (e.g. using a CCD) is a method of reading of theoptical data written in the material.

In the absence of light, the M-state molecules gradually reverted to thebasal color in about 1-2 minutes. When the M-state molecules in theexcited (yellow) state are exposed briefly (less than about a second) toa reading light, for example, purple light (e.g. from a halogen lampwith a 456 nm cut-off filter) or blue light (e.g. from a blue lightemitting diode (LED)), the color of the excited molecules are revertedto the basal color. This corresponds to rapid erasing of the opticalsignal imprinted in the film. These cycles can be repeated, therebyproviding a writable, readable, erasable, and rewritable opticalmaterial. One advantage of the use of proteorhodopsin and/orbacteriorhodopsin molecules is the ability of the molecules to withstandmultiple read and write cycles without photobleaching (loss of signal).

The present invention provides an optical data storage materialscomprising the solid material comprising various proteorhodopsins and/orbacteriorhodopsins as described above, wherein data are writtendifferentially by actinic light (writing light) of various wavelengthsand optical signals are read differentially by reading light of variouswavelengths. Optical signals are read differentially by determining thedecrease of B-state molecules of each of said proteorhodopsin orbacteriorhodopsin molecule. Alternatively, optical signals are readdifferentially by determining the absorbance of light at the M-statemaximum absorption wavelength by each of said proteorhodopsin orbacteriorhodopsin molecules.

Each of the multiple information bits within each domain is capable ofbeing selectively written in a process induced by differing wavelengthsof actinic light. Each actinic light has a wavelength that is uniquelyassociated with the maximum absorbance wavelength of a particularproteorhodopsin or bacteriorhodopsin molecule located within the sameaddressable domain. Each actinic light has a unique wavelength, which issuitable to cause predominantly only one particular proteorhodopsin orbacteriorhodopsin molecule to excite from its B-state to M-state.Accordingly, a single type of proteorhodopsin or bacteriorhodopsinmolecule (a bit), which has a particular maximum absorption wavelength,is written at by an actinic light of a particular wavelength. Byindependently addressing each of the various proteorhodopsin and/orbacteriorhodopsin molecules, data is written independently, thus thecapacity of data storage is increased due to the presence of multiplephotochromic molecules in the same addressable domain.

Each of the multiple information bits within each addressable domain isalso capable of being selectively read in a process induced by differingwavelengths of reading (probing) light.

One advantage to utilize a mixture of photochromic molecules is theability to store multiple bits within the same physical space (i.e.increased density). This ability to increase the number of bits within aspecific space allows for quaternary, octal or hexadecimal data storage.For example, a molecule can be in either a B or M state, represented bya 1 or a 0, which is a binary data storage scale. For each molecule, 2bits of data are stored. The number of recording modes occupying thesame addressable space is expressed as 2^(n), where n is the number ofphotochromic molecules that can be individually written or read. Forexample, the ability to put molecules with two different reading orwriting wavelengths in the same addressable space allows for quaternary(2²) storage. The ability to put molecules with three different readingor writing wavelengths in the same addressable space allows octal (2³)storage.

In one embodiment of the invention, an optical data storage material isswept under multiple read stations, each of which has a uniquewavelength of actinic light or reading light. In another embodiment ofthe invention, an optical data storage material is written or readsimultaneously by multiple wavelengths of actinic light or readinglight, each wavelength corresponds to a uniqueproteorhodopsin/bacteriorhodopsin molecule.

The image formed on the material such as a film can represent any kindof information that can be formed as individual data points on themolecules within the photochromic materials in the film. The larger thenumber of individual molecules in the photochromic mixture, the greaterthe optical density (O.D.), and the greater the signal of images storedtherein.

The present invention provides an optical data storage device comprisingone or more light sources and an optical data information carrier asdescribed above. In one embodiment, the one or more light sources emitindependently actinic writing light of different wavelengths to convertsaid various photochromic materials from a basal state to a M-state. Inanother embodiment, the one or more light sources emit reading light ofdifferent wavelengths to convert said various photochromic materialsfrom the M-state into the basal state.

The present invention further provides a fraud-proof data carriercomprising a solid material comprising an immobilized mixture ofdifferent proteorhodopsins/bacteriorhodopsins described above, whereinsaid different proteorhodopsins/bacteriorhodopsins have absorptionspectra that do not overlap significantly. The material containingimmobilized proteorhodopsin can be spread, sprayed, solidified, printed,deposited or dried on the surface of glass, paper, fabric materials,plastic material, metal surface or mineral surface for use as afraud-proof data carrier. The materials containing immobilizedproteorhodopsin/bacteriorhodopsin molecules can also be shaped in a moldto form the three-dimensional fraud-proof data carrier.

For example, the solid material is deposited on products such asbanknotes, documents, ID cards, passports, drivers' licenses, keycards,checks, securities, stickers, foils, containers, product packingmaterials etc., to guarantee the authenticity of the products. Whenproteorhodopsin/bacteriorhodopsin is exposed to light of excitationwavelength, it is excited to an activated M-state and changes to ayellow color. The color is reverted to its basal color eitherspontaneously with time or by exposing the material to a second light.For example, the proteorhodopsin molecule is excited by a yellow lightor a green light to change color from red or purple to yellow; the colorchange is erased spontaneously or by illuminating the material withpurple or blue light. The color change of proteorhodopsin orbacteriorhodopsin is reversible between the basal state and M-state,which provides protection against falsification. The write-read-erasecycle can be repeated multiple times without any observable change inthe property of the material. Conventional inks based on pigments ororganic dyes cannot mimic this color change. The color change featuremakes the proteorhodopsin and/or bacteriorhodopsin containing materialsdifficult for counterfeiters to mimic. By using a mixture of differentproteorhodopsin and/or bacteriorhodopsin molecules, multiple basalcolors can be provided in the same addressable domain in security ink ordocuments, which makes it even more difficult to counterfeit. Thesedifferent colored proteorhodopsin and/or bacteriorhodopsin molecules canbe any combination of all-trans-retinal-containing proteorhodopsins,retinal analog-containing proteorhodopsins, all-trans-retinal-containingbacteriorhodopsin, and retinal analog-containing bacteriorhodopsin, aslong as they have different maximum absorbance wavelengths.

Security Ink

The present invention further provides security ink comprising a mixtureof different proteorhodopsins and/or bacteriorhodopsins as describedabove and one or more hydrophilic polymers in a liquid form; thepolymers and the proteorhodopsins and/or bacteriorhodopsins form ahomogeneous phase. The security ink solidifies or dries after it isapplied onto a surface; and proteorhodopsins and/or bacteriorhodopsinsare immobilized onto a localized region where the ink is applied toprovide the security features. The security ink in general iswater-based, which is dried or solidified in air and forms a film. Thedrying or solidification of the ink results from loss of solvent,polymerization, or curing.

The security ink is prepared by combining a mixture of proteorhodopsinsand/or bacteriorhodopsins with one or more hydrophilic polymers in anaqueous solution to form a homogeneous solution. Optionally, auxiliaryagents such as binders, UV absorbers or dyes are included in thesecurity ink. Binders increase the binding or adhesion of photochromicmaterial to the surface that the ink is applied upon. Binders useful forthe present invention include gum arabic, polyvinyl acetate, polyvinylalcohol, and polyethylene glycol. UV absorbers protect the photochromicmaterial from UV damage and increase the UV-resistance of the securityink. UV absorbers include benzophenone, hydroxynaphthoquinone,phenylbenzoxazole, cinnamic acid esters, sulfonamide and aminobenzoicacid esters. Dyes modify the visual appearance of the ink. Otheradditives that may be included in the security ink are opticalbrighteners, driers, anti-skinning agents, thixotropy promoters, waxes,plasticizers, surfactants, defoaming agents and biocides. Thehydrophilic polymers can be any water-compatible polymers in which amixture of proteorhodopsins and/or bacteriorhodopsins can be evenlydispersed to form a homogeneous solution. Preferably, the solutioncontaining proteorhodopsins and/or bacteriorhodopsins and the polymerscan be dried in air quickly (within a minute or less) and form a filmthat allows efficient light absorption to excite the basal state of thephotochromic materials. In one embodiment of the invention, thehydrophilic polymer is gum arabic, polyvinylalcohol, polyvinyl acetate,polyethyleneglycol or polyvinyl pyrrolidone

In one embodiment of the invention, the security ink can be printed onpaper, foil, glass, metal surface, or plastic.

In another embodiment of the invention, the security ink can be appliedvia screen-printing or ink jet printing onto a document. At ambientconditions and usual room-light illuminations, the area printed from thesecurity ink appears, for example, purple or red color depending on thebasal state of the molecules within the mixture of photochromicmaterial. However, an increase of the light intensity would lead to arapid change of the color to yellow (M-state). Therefore, unauthorizedcopies produced by digital scanning or photocopying of documents printedwith security ink are easy to be distinguished from the authenticdocument. Conventional inks based on pigments or organic dyes cannotmimic this color change. The color change feature makes the photochromicmaterial difficult for counterfeiters to mimic.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES Example 1 Temporary Data Storage

Purified bacteriorhodopsin (BR) from Halobacterium salinarum, mutantD96N (Zeisel and Hampp, J. Phys. Chem., 1992. 96:7788-7792), 0.89 mg,was dissolved into 110 μl water with sonication. To this, 89 μl of 20%polyethyleneimine was added drop wise with vortex mixing to yield aclear purple solution. To 110 μl solution of 8 mg/ml purifiedproteorhodopsin mutant, Bac31A8/1108Q (U.S. Application Publication No.2005-0095605), 89 μl of 20% polyethyleneimine was added drop wise withvortex mixing to yield a clear red solution. These two purple and readsolutions were mixed together as a BR/PR PEI mixture. An immobilized gelof the BR/PR mixture was prepared by mixing 400 μl of the BR/PR PEImixture, 246 μl of water, and 20 μl of a 2.5% solution of high molecularweight polyvinylacetate. This mixture was warmed to about 50° C. andmixed with 94 μl of a 2% solution of purified agar at 55° C. Thesolution was mixed on a vortex mixer and pipetted into a 1.0 ml plasticcuvette. The solution was allowed to cool to room temperature.

Three small keychain LED lights having a maximum intensity at 400 nm,510 nm, and 640 nm respectively, were used for the state switching. Thecuvette containing the PR/BR gel was placed into the spectrophotometerand illuminated by the designated LED light (400 nm, 510 nm, or 400 nmfollowed by 640 nm) for several seconds. The designated LED light wasplaced over the cuvette for illumination for about 2 seconds and thenremoved. Spectra were then recorded on an HP 8452 Diode Arrayspectrophotometer in a dark room (FIGS. 1 and 2).

After the mixture containing BR and PR was illuminated with a violetlight (400 nm), both BR and PR were at basal (B) state, which absorb at570 nm and 520 nm respectively. See Spectra 1 and 5 in FIGS. 1 and 2.

Red light (640 nm) was capable of exciting BR from the B state to the Mstate but had no effect on exciting PR from the B sate. After themixture containing BR and PR was illuminated with a violet light (400nm) followed by a red light (640 nm), BR photocyled to the B state andthen to the M state, and PR photocycled to the B state and remained atthe B state. See Spectra 3 and 6 in FIGS. 1 and 2.

After the mixture containing BR and PR was illuminated with a greenlight (510 nm), both BR and PR were excited to the M state. See Spectra2 and 4 in FIGS. 1 and 2.

A simple model to evaluate these spectra is the ratio of absorbance at410 nm/560 nm

TABLE 1 Illumination sequence A₄₁₀/A₅₆₀ Ratio 400 nm 1.11 Spectrum 1 510nm 2.59 Spectrum 2 400 nm, followed by 640 nm 1.52 Spectrum 3 510 nm2.69 Spectrum 4 400 nm 1.08 Spectrum 5 400 nm, followed by 640 nm 1.55Spectrum 6The A₄₁₀/A₅₆₀ ratio can be considered a “digital state value” asdescribed in Table 2.

TABLE 2 Illumination Digital Photocycle State sequence Ratio 410/560state value BR PR After 400 nm light 1.1 0 B B 400 nm followed by 1.5 1M B 640 nm light After 510 nm light 2.6 2 M M

The data from this experiment demonstrate that a mixture containing BRand PR has the ability to temporarily encode, with light, differentdigital states.

The resolution of the data density that could be achieved with properoptical equipment would be expected to be no less than that for BR filmsas BR is the largest component (1-3μ) in the mixture. PR is monomeric(about 4-5 nm), thus PR would not be expected to affect the resolutionsignificantly to the smallest addressable size.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the scope of theinvention.

1. A solid material comprising an immobilized mixture of one or morebacteriorhodopsins and one or more proteorhodopsins.
 2. The solidmaterial according to claim 1, wherein said proteorhodopsins areall-trans-retinal-containing proteorhodopsins and saidbacteriorhodopsins are all-trans-retinal-containing bacteriorhodopsins.3. The solid material according to claim 1, wherein all of saidbacteriorhodopsins and proteorhodopsins have absorption spectra that donot overlap significantly.
 4. A solid material comprising an immobilizedmixture of two or more proteorhodopsins, wherein all of saidproteorhodopsins have absorption spectra that do not overlapsignificantly.
 5. The solid material according to claim 1 or 4, whereinsaid proteorhodopsins are all-trans-retinal-containing proteorhodopsinsor retinal analog-containing proteorhodopsins.
 6. A solid materialcomprising an immobilized mixture of two or more bacteriorhodopsinsselected from the group consisting of all-trans-retinal-containingbacteriorhodopsins and retinal analog-containing bacteriorhodopsins, allof said bacteriorhodopsins have absorption spectra that do not overlapsignificantly.
 7. The solid material according to claim 1, 4, or 6,wherein said solid material comprises one or more hydrophilic polymersthat are capable of forming a homogeneous phase with saidproteorhodopsins or said bacteriorhodopsins prior to solidification to asolid form.
 8. The solid material according to claim 7, wherein saidhydrophilic polymer is selected from the group consisting of silica solgel, gelatin, polyvinylalcohol, polyacrylamide, agarose, agar, methylcellulose, polyvinyl acetate and polyvinyl pyrrolidone, and polyethyleneglycol.
 9. An optical information carrier comprising the solid materialaccording to claim 1, 4, or 6, wherein data are written differentiallyby actinic light of different wavelengths and optical signals are readdifferentially by reading light of different wavelengths.
 10. Theoptical information carrier according to claim 9, wherein the opticalsignals are read differentially by determining the decrease of B-statemolecules of each said proteorhodopsin or bacteriorhodopsin.
 11. Theoptical information carrier according to claim 9, wherein the opticalsignals are read differentially by determining the absorbance of lightat the M-state maximum absorption wavelength of each saidproteorhodopsin or bacteriorhodopsin.
 12. The optical informationcarrier according to claim 9, further comprising a substrate selectedfrom the group consisting of glass, paper, metal, fabric material,plastic material, and combination thereof, wherein said solid materialis deposited on said substrate.
 13. The optical information carrieraccording to claim 9, wherein said optical information carrier is afraud-proof optical data carrier or an optical data storage material.14. The optical information carrier according to claim 9, wherein saidoptical information carrier provides non-destructive writing and readingof data and are capable of being reuse.
 15. An optical data storagedevice comprising one or more light sources and an optical datainformation carrier according to claim 9, wherein said one or more lightsources emit independently actinic writing light of differentwavelengths to convert said proteorhodopsins or bacteriorhodopsins froma basal state to a M-state.
 16. The optical data storage deviceaccording to claim 15, wherein said one or more light sources emitreading light of different wavelengths to convert said proteorhodopsinsor bacteriorhodopsins from the M-state into the basal state.
 17. Asecurity ink comprising photochromic materials and one or morehydrophilic polymers, wherein said photochromic materials and thehydrophilic polymers form a homogeneous liquid phase, said inksolidifies or dries after application onto a surface, therebyimmobilizing said photochromic materials onto a specific location wherethe ink is applied, wherein said photochromic materials are selectedfrom the group consisting of all-trans retinal containingproteorhodopsins, retinal analog-containing proteorhodopsins, all-transretinal containing bacteriorhodopsins, and retinal analog-containingbacteriorhodopsins, all of said proteorhodopsins and bacteriorhodopsinshave different absorption spectra.
 18. The security ink according toclaim 17, wherein said hydrophilic polymer is gum arabica, polyvinylalcohol, polyvinyl acetate, polyethylene glycol or polyvinylpyrrolidone.